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Abstract

Adjusting the objective correction collar is a widely used approach to correct spherical aberrations (SA) in optical microscopy. In this work, we characterized and compared its performance with adaptive optics in the context of in vivo brain imaging with two-photon fluorescence microscopy. We found that the presence of sample tilt had a deleterious effect on the performance of SA-only correction. At large tilt angles, adjusting the correction collar even worsened image quality. In contrast, adaptive optical correction always recovered optimal imaging performance regardless of sample tilt. The extent of improvement with adaptive optics was dependent on object size, with smaller objects having larger relative gains in signal intensity and image sharpness. These observations translate into a superior performance of adaptive optics for structural and functional brain imaging applications in vivo, as we confirmed experimentally.

Fig. 3 Effects on image size and brightness of different aberration correction methods as a function of bead size. The (a) normalized lateral FWHM, (b) normalized axial FWHM, and (c) normalized intensity for beads of different diameters (0.2, 0.5, 1, and 2 µm) under a single ~170-µm-thick coverglass with a tilt angle of 5°. Black dots represent individual bead measurements. Colored dots and error bars are the mean and standard deviation, respectively. Normalization was performed with respect to values obtained with syscor.

Fig. 4In vivo TPEF structural imaging with spherical aberration or full adaptive optical correction in the brain of a Thy1-GFP line M mouse. The cranial window was tilted by 3.5° (a-f) or 5° (g-h). Maximum intensity projections over 20 µm in z at a central depth of 25 µm of images recorded with (a,g) CC and (b,h) CC + AO. Scale bar: 20 µm. Maximum intensity projection along the y-axis for the images recorded with (c) CC and (d) CC + AO. Scale bar: 20 µm. Axial-lateral images of a spine at a depth of 25 µm measured with (c) CC and (d) CC + AO. Scale bar: 1 µm. (a) and (b), (c) and (d), (e) and (f), and (g) and (h) are plotted on the same intensity scale, respectively. z: axial; x and y: lateral.